In order to extend capabilities in the study of the effect of amino acid sequence on protein function, a novel enzymatic resynthesis method was applied to several noncovalent fragment complexes. Substitution of water by partially nonaqueous solvents (e.g., 90% glycerol) in systems of limited proteolyses, thermodynamically stabilizes the peptide bond and allows resynthesis (30-50%). A noncovalent complex of ribonuclease S-protein and 4-Fl-His12-S-peptide was converted to a covalent form, allowing the study of catalysis at a low pH where the complex would have dissociated. A counterpart system where a peptide, 4-Fl-His119 (116-124) was chemically synthesized and complexed to ribonuclease-(1-115) provided additional information to suggest that both His12 and His119 are general catalytic acid bases. A two-fragment staphylococcal nuclease complex was "restitched" with the exclusion of loop residue, Lys49. This completely abolished activity, suggesting the importance of the integrity of the loop for the correct conformation at the active site. A cytochrome c complex and a prothrombin l complex were also restitched, demonstrating the potential for extended studies of covalent hybrids of these proteins.